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  • 1.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Divne, Anna-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Universal tag arrays in forensic SNP analysis.2005In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 297, p. 141-154Article in journal (Refereed)
    Abstract [en]

    Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.

  • 2.
    Andersson, Jan O
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Siv GE
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Genomic rearrangements during evolution of the obligate intracellular parasite Rickettsia prowazekii as inferred from an analysis of 52015 bp nucleotide sequence1997In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 143, no 8, p. 2783-2795Article in journal (Other academic)
    Abstract [en]

    In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome of Rickettsia prowazekii, a member of the alpha-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue of metK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures of Escherichia coli, Haemophilus influenzae and Bacillus subtilis have revealed several atypical gene organization patterns in the R. prowazekii genome. For example, R. prowazekii was found to have a unique arrangement of genes upstream of dnaA in a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that the R. prowazekii genome is a highly derived genome and provide examples of gene order structures that are unique for the Rickettsia.

  • 3.
    Andersson, Siv GE
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Zomorodipour, A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Jan O
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sicheritz-Ponten, T
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Alsmark, UCM
    Uppsala University.
    Podowski, RM
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Näslund, A Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Eriksson, Ann-Sofie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Winkler, HH
    Kurland, Charles G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The genome sequence of Rickettsia prowazekii and the origin of mitochondria1998In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 396, no 6707, p. 133-140Article in journal (Refereed)
    Abstract [en]

    We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of 'neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

  • 4.
    Björkman, Johanna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Samuelsson, Patrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium1999In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 31, no 1, p. 53-58Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.

  • 5.
    Bouakaz, Lamine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Bouakaz, Elli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Murgola, Emanuel J.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 7, p. 4548-4556Article in journal (Refereed)
    Abstract [en]

    It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide release by, in particular, the class I release factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (ΔL11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4-6-fold reductions in termination efficiency (kcat/Km) of RF1, but not of RF2, on ΔL11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.

  • 6. Budowle, B.
    et al.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Chakraborty, R.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Forensic analysis of the mitochondrial coding region and association to disease.2005In: International journal of legal medicine (Print), ISSN 0937-9827, E-ISSN 1437-1596, Vol. 119, no 5, p. 314-315Article in journal (Refereed)
  • 7. Desmet, Tom
    et al.
    Claeyssens, Marc
    Piens, Kathleen
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Nerinckx, Wim
    Synthesis and Evaluation of 2-Deoxy-2-amino-beta-cellobiosides as Cellulase Inhibitors2010In: Journal of carbohydrate chemistry, ISSN 0732-8303, E-ISSN 1532-2327, Vol. 29, no 4, p. 164-180Article in journal (Refereed)
    Abstract [en]

    The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining -glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido--cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI= 1 M).

  • 8.
    Divne, Anna-Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A DNA microarray system for forensic SNP analysis2005In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 154, no 2-3, p. 111-121Article in journal (Refereed)
    Abstract [en]

    Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.

  • 9.
    Divne, Anna-Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Martina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Calloway, Cassandra
    Reynolds, Rebecca
    Erlich, Henry
    Allen, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Forensic casework analysis using the HVI/HVII mtDNA linear array assay.2005In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 50, no 3, p. 548-554Article in journal (Refereed)
    Abstract [en]

    The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.

  • 10.
    Dong, Hengjiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Nilsson, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Growth rate regulation of 4.5 S RNA and M1 RNA, the catalytic subunit of Escherichia coli RNase P1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 261, p. 303-308Article in journal (Refereed)
    Abstract [en]

    We have studied the expression of 4.5 S RNA and Fv Il RNA, the catalytic subunit of Escherchia coli RNase P, under various growth conditions. Both RNA species increase in abundance as a function of growth rate. There are roughly 450 molecules of 4.5 S RNA and 80 molecules of M1 RNA per cell at 0.4 doubling per hour, and this is increased to 5300 and 1060 molecules per cell, respectively, at 2.7 doublings per hour. Deletion of both relA and spoT, the two genes that are responsible for synthesis of ppGpp, does not affect the rate of synthesis of either RNA species. However, deletion of fis renders the expression of 4.5 S RNA independent of growth rate, but has little effect on the expression of M1 RNA. These data suggest that the expression of both 4.5 S RNA and M1 RNA genes are growth-rate regulated, but not through the same mechanism. The growth-rate dependent accumulation of 4.5 S RNA depends on FIS-mediated trans-activation, whereas that of M1 RNA is not governed by ppGpp or by FIS.

  • 11.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Crist, Colin G
    Nakamura, Yoshikazu
    Translation Termination, the Prion [Psi+], and Ribosomal Recycling2007In: Translational Control in Biology and Medicine / [ed] Michael B. Mathews, Nahum Sonenberg & John W.B. Hershey, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press , 2007, p. 173-196Chapter in book (Other academic)
  • 12.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Sjöberg, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Problems of high dimension in molecular biology2003In: Proc. 19th GAMM Seminar Leipzig on High-dimensional problems: Numerical treatment and applications, Leipzig, Germany: Max Planck Institute for Mathematics in the Sciences , 2003, p. 21-30Conference paper (Refereed)
  • 13.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Paulsson, Johan
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mesoscopic kinetics and its applications in protein synthesis2005In: Systems Biology: Definitions and Perspectives / [ed] Lila Alberghina and H.V. Westerhoff, Heidelberg: Springer-Verlag GmbH , 2005, p. 95-118Chapter in book (Other academic)
    Abstract [en]

    Molecular biology emerged through unification of genetics and nucleic acid chemistry that took place with the discovery of the double helix (Watson and Crick 1953). Accordingly, molecular biology could be defined as the sum of all techniques used to perform genetic experiments by manipulating DNA. One consequence of the development of these techniques is large-scale sequencing of genomes from an ever increasing number of organisms. However, it became clear from this development that genetic information per se is not enough to grasp the most interesting functional and evolutionary aspects of cells and multi-cellular organisms. In fact, understanding how genotype leads to phenotype depends on concepts and techniques from areas that so far have been largely alien to molecular biological research, like physics, mathematics, and engineering. From the bits and pieces from these and other scientific fields new tools must be generated to make possible an understanding of the dynamic, adapting, and developing living systems that somehow take shape from the instructions given by their genomes. The growing total of these tools and their integration in experimental and theoretical approaches to understand complex biological processes in ways previously out of reach could be a way to define systems biology, in analogy with the above definition of molecular biology.

  • 14.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Modelling Approaches to Molecular Systems Biology2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Implementation and analysis of mathematical models can serve as a powerful tool in understanding how intracellular processes in bacteria affect the bacterial phenotype. In this thesis I have implemented and analysed models of a number of different parts of the bacterium E. coli in order to understand these types of connections. I have also developed new tools for analysis of stochastic reaction-diffusion models.

    Resistance mutations in the E. coli ribosomes make the bacteria less susceptible to treatment with the antibiotic drug erythromycin compared to bacteria carrying wildtype ribosomes. The effect is dependent on efficient drug efflux pumps. In the absence of pumps for erythromycin, there is no difference in growth between wildtype and drug target resistant bacteria. I present a model explaining this unexpected phenotype, and also give the conditions for its occurrence.

    Stochastic fluctuations in gene expression in bacteria, such as E. coli, result in stochastic fluctuations in biosynthesis pathways. I have characterised the effect of stochastic fluctuations in the parallel biosynthesis pathways of amino acids. I show how the average protein synthesis rate decreases with an increasing number of fluctuating amino acid production pathways. I further show how the cell can remedy this problem by using sensitive feedback control of transcription, and by optimising its expression levels of amino acid biosynthetic enzymes.

    The pole-to-pole oscillations of the Min-proteins in E. coli are required for accurate mid-cell division. The phenotype of the Min-oscillations is altered in three different mutants: filamentous cells, round cells and cells with changed membrane lipid composition. I have shown that the wildtype and mutant phenotypes can be explained using a stochastic reaction-diffusion model.

    In E. coli, the transcription elongation rate on the ribosmal RNA operon increases with increasing transcription initiation rate. In addition, the polymerase density varies along the ribosomal RNA operons. I present a DNA sequence dependent model that explains the transcription elongation rate speed-up, and also the density variation along the ribosomal operons. Both phenomena are explained by the RNA polymerase backtracking on the DNA.

    List of papers
    1. Drug efflux pump deficiency and drug target resistance masking in growing bacteria
    Open this publication in new window or tab >>Drug efflux pump deficiency and drug target resistance masking in growing bacteria
    2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 20, p. 8215-8220Article in journal (Refereed) Published
    Abstract [en]

    Recent experiments have shown that drug efflux pump deficiency not only increases the susceptibility of pathogens to antibiotics, but also seems to "mask" the effects of mutations, that decrease the affinities of drugs to their intracellular targets, on the growth rates of drug-exposed bacteria. That is, in the presence of drugs, the growth rates of drug-exposed WT and target mutated strains are the same in a drug efflux pump deficient background, but the mutants grow faster than WT in a drug efflux pump proficient background. Here, we explain the mechanism of target resistance masking and show that it occurs in response to drug efflux pump inhibition among pathogens with high-affinity drug binding targets, low cell-membrane drug-permeability and insignificant intracellular drug degradation. We demonstrate that target resistance masking is fundamentally linked to growth-bistability, i.e., the existence of 2 different steady state growth rates for one and the same drug concentration in the growth medium. We speculate that target resistance masking provides a hitherto unknown mechanism for slowing down the evolution of target resistance among pathogens.

    Keywords
    antibiotic resistance, efflux pump inhibition, macrolides
    National Category
    Biological Sciences
    Research subject
    Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-104045 (URN)10.1073/pnas.0811514106 (DOI)000266209000025 ()19416855 (PubMedID)
    Available from: 2009-05-27 Created: 2009-05-27 Last updated: 2017-12-13Bibliographically approved
    2. Not competitive enzyme inhibitors revisited
    Open this publication in new window or tab >>Not competitive enzyme inhibitors revisited
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

     

     

    Identifiers
    urn:nbn:se:uu:diva-133222 (URN)
    Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2011-01-13
    3. Deleterious effects of fluctuations in parallel metabolic  pathways
    Open this publication in new window or tab >>Deleterious effects of fluctuations in parallel metabolic  pathways
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

     

     

     

     

     

    Identifiers
    urn:nbn:se:uu:diva-133221 (URN)
    Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2011-01-13
    4. Noise-induced Min phenotypes in E. coli
    Open this publication in new window or tab >>Noise-induced Min phenotypes in E. coli
    2006 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 2, no 6, p. 637-648Article in journal (Refereed) Published
    Abstract [en]

    The spatiotemporal oscillations of the Escherichia coli proteins MinD and MinE direct cell division to the region between the chromosomes. Several quantitative models of the Min system have been suggested before, but no one of them accounts for the behavior of all documented mutant phenotypes. We analyzed the stochastic reaction-diffusion kinetics of the Min proteins for several E. coli mutants and compared the results to the corresponding deterministic mean-field description. We found that wild-type (wt) and filamentous (ftsZ(-)) cells are well characterized by the mean-field model, but that a stochastic model is necessary to account for several of the characteristics of the spherical (rodA(-)) and phospathedylethanolamide-deficient (PE-) phenotypes. For spherical cells, the mean-field model is bistable, and the system can get trapped in a non-oscillatory state. However, when the intrinsic noise is considered, only the experimentally observed oscillatory behavior remains. The stochastic model also reproduces the change in oscillation directions observed in the spherical phenotype and the occasional gliding of the MinD region along the inner membrane. For the PE- mutant, the stochastic model explains the appearance of randomly localized and dense MinD clusters as a nucleation phenomenon, in which the stochastic kinetics at low copy number causes local discharges of the high MinD(ATP) to MinD(ADP) potential. We find that a simple five-reaction model of the Min system can explain all documented Min phenotypes, if stochastic kinetics and three-dimensional diffusion are accounted for. Our results emphasize that local copy number fluctuation may result in phenotypic differences although the total number of molecules of the relevant species is high.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-18951 (URN)10.1371/journal.pcbi.0020080 (DOI)000239494000016 ()16846247 (PubMedID)
    Available from: 2006-11-24 Created: 2006-11-24 Last updated: 2017-12-08Bibliographically approved
    5. Stochastic reaction-diffusion kinetics in the microscopic limit
    Open this publication in new window or tab >>Stochastic reaction-diffusion kinetics in the microscopic limit
    2010 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 46, p. 19820-19825Article in journal (Refereed) Published
    Abstract [en]

    Quantitative analysis of biochemical networks often requires consideration of both spatial and stochastic aspects of chemical processes. Despite significant progress in the field, it is still computationally prohibitive to simulate systems involving many reactants or complex geometries using a microscopic framework that includes the finest length and time scales of diffusion-limited molecular interactions. For this reason, spatially or temporally discretized simulations schemes are commonly used when modeling intracellular reaction networks. The challenge in defining such coarse-grained models is to calculate the correct probabilities of reaction given the microscopic parameters and the uncertainty in the molecular positions introduced by the spatial or temporal discretization. In this paper we have solved this problem for the spatially discretized Reaction-Diffusion Master Equation; this enables a seamless and physically consistent transition from the microscopic to the macroscopic frameworks of reaction-diffusion kinetics. We exemplify the use of the methods by showing that a phosphorylation-dephosphorylation motif, commonly observed in eukaryotic signaling pathways, is predicted to display fluctuations that depend on the geometry of the system.

     

     

    Keywords
    diffusion-limited, mesoscopic, master equation, Smoluchowski, spatial
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-133220 (URN)10.1073/pnas.1006565107 (DOI)000284261800042 ()21041672 (PubMedID)
    Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2017-12-12Bibliographically approved
    6. Stochastic reaction-diffusion simulation with MesoRD.
    Open this publication in new window or tab >>Stochastic reaction-diffusion simulation with MesoRD.
    2005 (English)In: Bioinformatics, ISSN 1367-4803, Vol. 21, no 12, p. 2923-4Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-74098 (URN)15817692 (PubMedID)
    Available from: 2005-08-29 Created: 2005-08-29 Last updated: 2011-01-13
    7. Varying rate of RNA chain elongation during rrn transcription in Escherichia coli.
    Open this publication in new window or tab >>Varying rate of RNA chain elongation during rrn transcription in Escherichia coli.
    2009 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 11, p. 3740-3746Article in journal (Refereed) Published
    Abstract [en]

    The value of the rRNA chain elongation rate in bacteria is an important physiological parameter, as it affects not only the rRNA promoter activity but also the free-RNA polymerase concentration and thereby the transcription of all genes. On average, rRNA chains elongate at a rate of 80 to 90 nucleotides (nt) per s, and the transcription of an entire rrn operon takes about 60 s (at 37 degrees C). Here we have analyzed a reported distribution obtained from electron micrographs of RNA polymerase molecules along rrn operons in E. coli growing at 2.5 doublings per hour (S. Quan, N. Zhang, S. French, and C. L. Squires, J. Bacteriol. 187:1632-1638, 2005). The distribution exhibits two peaks of higher polymerase density centered within the 16S and 23S rRNA genes. An evaluation of this distribution indicates that RNA polymerase transcribes the 5' leader region at speeds up to or greater than 250 nt/s. Once past the leader, transcription slows down to about 65 nt/s within the 16S gene, speeds up in the spacer region between the 16S and 23S genes, slows again to about 65 nt/s in the 23S region, and finally speeds up to a rate greater than 400 nt/s near the end of the operon. We suggest that the slowing of transcript elongation in the 16S and 23S sections is the result of transcriptional pauses, possibly caused by temporary interactions of the RNA polymerase with secondary structures in the nascent rRNA.

     

    National Category
    Biological Sciences
    Research subject
    Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-104047 (URN)10.1128/JB.00128-09 (DOI)000266041300035 ()19329648 (PubMedID)
    Available from: 2009-05-27 Created: 2009-05-27 Last updated: 2017-12-13Bibliographically approved
    8. Rate of  ribosomal RNA transcription modulated by the rrn operon  sequence and RNA polymerase interaction
    Open this publication in new window or tab >>Rate of  ribosomal RNA transcription modulated by the rrn operon  sequence and RNA polymerase interaction
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

     

     

     

    Identifiers
    urn:nbn:se:uu:diva-133223 (URN)
    Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2011-01-13
  • 15.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Sjöberg, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stochastic reaction-diffusion kinetics in the microscopic limit2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 46, p. 19820-19825Article in journal (Refereed)
    Abstract [en]

    Quantitative analysis of biochemical networks often requires consideration of both spatial and stochastic aspects of chemical processes. Despite significant progress in the field, it is still computationally prohibitive to simulate systems involving many reactants or complex geometries using a microscopic framework that includes the finest length and time scales of diffusion-limited molecular interactions. For this reason, spatially or temporally discretized simulations schemes are commonly used when modeling intracellular reaction networks. The challenge in defining such coarse-grained models is to calculate the correct probabilities of reaction given the microscopic parameters and the uncertainty in the molecular positions introduced by the spatial or temporal discretization. In this paper we have solved this problem for the spatially discretized Reaction-Diffusion Master Equation; this enables a seamless and physically consistent transition from the microscopic to the macroscopic frameworks of reaction-diffusion kinetics. We exemplify the use of the methods by showing that a phosphorylation-dephosphorylation motif, commonly observed in eukaryotic signaling pathways, is predicted to display fluctuations that depend on the geometry of the system.

     

     

  • 16.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Deleterious effects of fluctuations in parallel metabolic  pathwaysManuscript (preprint) (Other academic)
    Abstract [en]

     

     

     

     

     

  • 17.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Rate of  ribosomal RNA transcription modulated by the rrn operon  sequence and RNA polymerase interactionManuscript (preprint) (Other academic)
    Abstract [en]

     

     

     

  • 18.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lovmar, Martin
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Not competitive enzyme inhibitors revisitedManuscript (preprint) (Other academic)
    Abstract [en]

     

     

  • 19.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Nilsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Tenson, Tanel
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Drug efflux pump deficiency and drug target resistance masking in growing bacteria2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 20, p. 8215-8220Article in journal (Refereed)
    Abstract [en]

    Recent experiments have shown that drug efflux pump deficiency not only increases the susceptibility of pathogens to antibiotics, but also seems to "mask" the effects of mutations, that decrease the affinities of drugs to their intracellular targets, on the growth rates of drug-exposed bacteria. That is, in the presence of drugs, the growth rates of drug-exposed WT and target mutated strains are the same in a drug efflux pump deficient background, but the mutants grow faster than WT in a drug efflux pump proficient background. Here, we explain the mechanism of target resistance masking and show that it occurs in response to drug efflux pump inhibition among pathogens with high-affinity drug binding targets, low cell-membrane drug-permeability and insignificant intracellular drug degradation. We demonstrate that target resistance masking is fundamentally linked to growth-bistability, i.e., the existence of 2 different steady state growth rates for one and the same drug concentration in the growth medium. We speculate that target resistance masking provides a hitherto unknown mechanism for slowing down the evolution of target resistance among pathogens.

  • 20. Gao, Haixiao
    et al.
    Zhou, Zhihong
    Rawat, Urmila
    Huang, Chenhui
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Bouakaz, Lamine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Chernhoe
    Cheng, Zhihong
    Liu, Yuying
    Zavialov, Andrey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Gursky, Richard
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Song, Haiwei
    RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors2007In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 129, no 5, p. 929-941Article in journal (Refereed)
    Abstract [en]

    During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3•GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu•GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3•GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.

  • 21. Gao, Ning
    et al.
    Zavialov, Andrey V.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 374, no 5, p. 1345-1358Article in journal (Refereed)
    Abstract [en]

    After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5′-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-Å (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S·EF-G·guanosine 5′-[(βγ)-imido]triphosphate·RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5′-[(βγ)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.

  • 22. Gruic, Mirjana
    et al.
    Braga, Tiago
    Lukinius, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Eloranta, Maija-Leena
    Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Knight, Stefan D.
    Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Pejler, Gunnar
    Åbrink, Magnus
    Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 39, p. 33411-33418Article in journal (Refereed)
    Abstract [en]

    Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.

  • 23.
    Hauryliuk, Vasili
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Hansson, Sebastian
    Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Co-factor dependent conformational switching of GTPases2008In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Biophysical J, ISSN 0006-3495, Vol. 95, no 4, p. 1704-1715Article in journal (Refereed)
    Abstract [en]

    This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP gamma S.

  • 24.
    Hauryliuk, Vasili
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mitkevich, Vladimir A
    Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
    Eliseeva, Natalia A
    Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
    Petrushanko, Irina Yu
    Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Makarov, Alexander A
    Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
    The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form.2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 41, p. 15678-83Article in journal (Refereed)
    Abstract [en]

    Translocation of the tRNA x mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4 degrees C, 20 degrees C, and 37 degrees C. The binding affinity of EF-G is higher to GDP than to GTP at 4 degrees C, but lower at 37 degrees C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the gamma-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an "activity chimera," with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its "ratcheted state," with hybrid tRNA binding sites.

  • 25.
    Henriksson, Niklas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Poly(A)-specific Ribonuclease (PARN): Structural and Functional Studies of Poly(A) Recognition and Degradation2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Regulation of mRNA degradation is a powerful way for the cell to regulate gene expression. A critical step in eukaryotic mRNA degradation is the removal of the poly(A) tail at the 3'-end of the mRNA. Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. In addition to the exonuclease domain, PARN harbors two RNA-binding domains, a classical RNA recognition motif (RRM) and an R3H-domain. In this project we have studied mechanisms by which PARN specifically recognizes and degrades poly(A).

    We investigated the RNA binding properties of PARN by using electrophoretic mobility shift assays and filter-binding analysis and we could show that PARN binds poly(A) with high affinity and specificity. Furthermore, we showed that the RRM and R3H domains of PARN separately could bind to poly(A).

    To investigate specificity for and recognition of poly(A) in the active site of PARN, we performed a kinetic analysis on a repertoire of trinucleotide substrates in vitro. We showed that PARN harbors affinity for adenosines in the active site and that both the penultimate and the 3' end located nucleotide play an important role for providing adenosine-specificity in the active site of PARN.

    Moreover, we solved a crystal structure of PARN in complex with m7GpppG cap analogue and showed that the cap binding and active sites overlap both structurally and functionally. By mutational analysis we identified residues in the active site that specifically recognize adenosines. Furthermore, biochemical data showed that the adenosine specificity in the active site is lost when Mn2+ is used instead of Mg2+ as divalent metal ion.

    Taken together, these results demonstrate that both RNA-binding properties of the RRM and R3H-domains in addition to base recognition in the active site contributes to PARN poly(A)-specificity.

     

     

    List of papers
    1. A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties
    Open this publication in new window or tab >>A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties
    Show others...
    2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 45, p. 32902-32911Article in journal (Refereed) Published
    Abstract [en]

    Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-97049 (URN)10.1074/jbc.M702375200 (DOI)000250625400039 ()17785461 (PubMedID)
    Available from: 2008-04-18 Created: 2008-04-18 Last updated: 2017-12-14Bibliographically approved
    2. Crystal Structure of PARN in Complex with Cap Analogue
    Open this publication in new window or tab >>Crystal Structure of PARN in Complex with Cap Analogue
    Show others...
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-97050 (URN)
    Available from: 2008-04-18 Created: 2008-04-18 Last updated: 2010-01-13Bibliographically approved
    3. Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease
    Open this publication in new window or tab >>Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease
    Show others...
    2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 1, p. 163-170Article in journal (Refereed) Published
    Abstract [en]

    Poly(A)-specific ribonuclease (PARN) is a mammalian 3′-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), ∼10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.

    Keywords
    mRNA deadenylation, poly(A)-specific ribonuclease, active site, trinucleotides, poly(A)-specificity, poly(U)-specificity
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-100494 (URN)10.1074/jbc.M109.043893 (DOI)000273070100017 ()19901024 (PubMedID)
    Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2017-12-13Bibliographically approved
    4. Poly(A)-specific ribonuclease (PARN) loses specificity for recognition of adenosine residues in the presence of Mn2+
    Open this publication in new window or tab >>Poly(A)-specific ribonuclease (PARN) loses specificity for recognition of adenosine residues in the presence of Mn2+
    (English)Manuscript (Other academic)
    Abstract [en]

    Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. PARN is a member of the DEDD superfamily of exonucleases and is dependent on divalent metal ions for its activity. Here, we have investigated how PARN poly(A) specificity can be modulated by using Mn2+ instead of Mg2+ as the divalent metal ion. In the presence of Mn2+ PARN lost specificity for poly(A) degradation. By using homotrinucleotides as substrates we could demonstrate that the loss in specificity was an intrinsic property of the active site in the presence of Mn2+ and not caused by changes in the RNA binding properties of PARN. We conclude that Mn2+ can be used as a probe to understand mechanisms behind poly(A) specificity in the active site of PARN.

    Keywords
    mRNA degradation, deadenylation, Poly (A)-specific ribonuclease, active site, poly(A)-specificity, Mn2+
    Identifiers
    urn:nbn:se:uu:diva-100497 (URN)
    Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2010-01-14
    5. RNA binding properties of the R3H domain of poly(A)-specific ribonuclease (PARN)
    Open this publication in new window or tab >>RNA binding properties of the R3H domain of poly(A)-specific ribonuclease (PARN)
    (English)Manuscript (Other academic)
    Abstract [en]

    Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. PARN harbors, in addition to a well characterized RNA-recognition motif (RRM), another putative RNA-binding motif: the R3H-domain. R3H-domains have previously been found in a diverse range of proteins involved in single stranded nucleic acid binding suggesting that the domain is a nucleic acid interacting motif. However, its RNA binding properties has not been experimentally verified. Here, we show that the R3H domain of PARN binds poly(A) and poly(U). Therefore, our study verifies that the R3H domain binds RNA and suggests that the R3H-domain of PARN could play a role in recognizing poly(A).

    Keywords
    mRNA degradation, deadenylation, Poly (A)-specific ribonuclease, poly(A)-specificity, R3H-domain
    Identifiers
    urn:nbn:se:uu:diva-100499 (URN)
    Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2010-01-14
  • 26.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Bouakaz, Elli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The kinetics of ribosomal peptidyl transfer revisited2008In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 30, no 5, p. 589-598Article in journal (Refereed)
    Abstract [en]

    The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

  • 27.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Rate and accuracy of bacterial protein synthesis revisited2008In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 11, no 2, p. 141-147Article, review/survey (Refereed)
    Abstract [en]

    Our understanding of the accuracy of tRNA selection on the messenger RNA programmed ribosome has recently increased dramatically because of high-resolution crystal structures of the ribosome, cryo-electron microscopy reconstructions of its functional complexes, and fast kinetics experiments. Application of single-molecule spectroscopy with fluorescence resonance energy transfer to studies of tRNA selection by the ribosome has also provided new, albeit controversial, insights. Interestingly, when the fundamental trade-off between rate and accuracy in substrate-selective biosynthetic reactions is taken into account, some aspects of the current models of ribosome function appear strikingly suboptimal in the context of growing bacterial cells.

  • 28.
    Larsson, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hinas, Andrea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ardell, David H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Söderbom, Fredrik
    De novo search for non-coding RNA genes in the AT-rich genome of Dictyostelium discoideum: Performance of Markov-dependent genome feature scoring2008In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 18, no 6, p. 888-899Article in journal (Refereed)
    Abstract [en]

    Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.

  • 29.
    Li, Wen
    et al.
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Agirrezabala, Xabier
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Lei, Jianlin
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Bouakaz, Lamine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Brunelle, Julie L
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Ortiz-Meoz, Rodrigo F
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Green, Rachel
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 24, p. 3322-3331Article in journal (Refereed)
    Abstract [en]

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.

  • 30.
    Lovmar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Nilsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lukk, Eliisa
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Vimberg, Vladimir
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Tenson, Tanel
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Erythromycin resistance by L4/L22 mutations and resistance masking by drug efflux pump deficiency2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 6, p. 736-744Article in journal (Refereed)
    Abstract [en]

    We characterized the effects of classical erythromycin resistance mutations in ribosomal proteins L4 and L22 of the large ribosomal subunit on the kinetics of erythromycin binding. Our data are consistent with a mechanism in which the macrolide erythromycin enters and exits the ribosome through the nascent peptide exit tunnel, and suggest that these mutations both impair passive transport through the tunnel and distort the erythromycin-binding site. The growth-inhibitory action of erythromycin was characterized for bacterial populations with wild-type and L22-mutated ribosomes in drug efflux pump deficient and proficient backgrounds. The L22 mutation conferred reduced erythromycin susceptibility in the drug efflux pump proficient, but not deficient, background. This 'masking' of drug resistance by pump deficiency was reproduced by modelling with input data from our biochemical experiments. We discuss the general principles behind the phenomenon of drug resistance 'masking', and highlight its potential importance for slowing down the evolution of drug resistance among pathogens.

  • 31.
    Lovmar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Vimberg, Vladimir
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Lukk, Eliisa
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Nilsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Tenson, Tanel
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Cis-acting resistance peptides reveal dual ribosome inhibitory action of the macrolide josamycin2009In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 91, no 8, p. 989-995Article in journal (Refereed)
    Abstract [en]

    Macrolide antibiotics block the entrance of nascent peptides to the peptide exit tunnel of the large ribosomal subunit. Expression of specific cis-acting peptides confers low-level macrolide-resistance. We show that, in the case of josamycin, peptide expression does not eject josamycin from the ribosome, implying a peptide resistance mechanism different from that previously suggested for erythromycin. We find dipeptide formation and dipeptidyl-tRNA drop-off in the presence of josamycin to be much slower during translation of resistance than of control mRNAs. We demonstrate low-level josamycin resistance by over-expression of peptidyl-tRNA hydrolase. These findings suggest dual growth-inhibitory action of josamycin by (i) direct inhibition of peptide-elongation and (ii) indirect inhibition of peptide-elongation through rapid peptidyl-tRNA drop-off, leading to depletion of tRNA isoacceptors available for protein synthesis. We propose that josamycin resistance peptide expression brings ribosomes into a "quarantine" state with small drop-off rate, thereby eliminating the josamycin dependent depletion of tRNA isoacceptors in the protein-synthesis-active state.

  • 32.
    Martinez, Javier